The present invention is directed to the use of the extract of Ginkgo biloba leaves or isolated ginkgolide B (GKB), a component of the extract of Ginkgo biloba leaves in a method for decreasing the expression of peripheral-type benzodiazepine receptor (PBR) in cells of a patient in need thereof. Further, the present invention is directed to the use of the extract of Ginkgo biloba leaves or isolated GKB in a method for decreasing the proliferation of cancer cells in a patient in need thereof. More particularly, the present invention is directed to the use of the extract of Ginkgo biloba leaves or isolated GKB in a method of decreasing cancer cell proliferation in a patient in need thereof wherein said cancer cell is human breast cancer cells. Even more particularly, the present invention is directed to the use of the extract of Ginkgo biloba leaves or isolated GKB in a method of decreasing cancer cell proliferation in a patient in need thereof wherein said cancer cell are of the aggressive and invasive phenotype and expresses high levels of PBR in comparison to non-aggressive cancer cells.
In another aspect, the present invention is directed to the use of extract of Ginkgo biloba leaves to decrease the expression of thirty-five (35) gene products as is further detailed, hereinbelow.
It is preferred that a particular formulation of Ginkgo biloba leaves extract known as EGB 761® (a product of IPSEN, Paris, France) be a constituent in a composition or used in a method of the present invention.
Ginkgo biloba is one of the most ancient trees and extracts from its leaves have been used in traditional medicine for several hundred years. There are numerous studies describing the beneficial effects of Ginkgo biloba extracts on patients with disturbances in vigilance, memory, and cognitive functions associated with aging and senility, and on those with all types of dementias, mood changes, and the ability to cope with daily stressors. A standardized extract of Ginkgo biloba leaves, termed EGB 761®, has been used in most of these studies. This extract is also known to have cardioprotective effects (DeFeudis F. V. Ginkgo biloba extract (EGB 761®): from chemistry to clinic. Ullstein Medical, Wisbaden, Germany. 400 pp. 1998; Tosaki, A., Droy-Lefaix, M. T., Pali, T., and Das, D. K., Free Rad. Biol. Med., 14: 361–370, 1993). These effects have been attributed, at least in part, to the free radical scavenging properties of EGb761®, probably due to the presence of flavonoid or terpenoid constituents in the extract. Recent in vivo and in vitro studies demonstrated that the terpene constituents of EGB 761®, ginkgolides and bilobalide, have anti-oxidant properties (Pietri, S., Maurelli, E., Drieu, K., and Culcasi, M., J. Mol. Cell. Cardiol., 29: 733–742, 1997; Yao, Z., Boujrad, N., Drieu, K., and Papadopoulos, V., Adv. Ginkgo Biloba Res. 7: 129–138, 1998). Other studies of EGB 761® have reported medicinal value of the product in the treatment of a variety of clinical disorders including cerebrovascular and peripheral vascular insufficiencies associated with aging and senility. See e.g., Ginkgo biloba Extract (EGB 761®) Pharmacological Activities and Clinical Applications, DeFeudis, F. V., Eds, Elsevier, 1991; and Ullstein Medical 1998, Ginkgo biloba extract (EGB 761®), Eds. Wiesbaden, DeFeudis, F. V. The extract contains 24% ginkgo-flavone glycosides, 6% terpene lactones (ginkgolides and bilobalide), about 7% proanthocyanidins and several other constituents. See Boralle, N., et al., In: Ginkgolides, Chemistry, Biology, Pharmacology and Clinical perspectives, Ed: Braquet, P., J. R. Prous Science Publishers, 1988.
Tumor progression is a multi-step process in which normal cells gradually acquire more malignant phenotypes, including the ability to invade tissues and form metastases, the primary cause of mortality in breast cancer. During this process, the “aberrant” expression of a number of gene products may be the cause or the result of tumorigenesis. Considering that the first step of tumor progression is cell proliferation, it can be proposed that tumorigenesis and malignancy are related to the proliferative potential of tumoral cells.
Studies in a number of tumors such as rat brain containing glioma tumors (Richfield, E. K. et al. (1988) Neurology 38:1255–1262), colonic adenocarcinoma and ovarian carcinoma (Katz, Y. et al. (1988) Eur. J. Pharmacol. 148:483–484 and Katz, Y. et al. (1990) Clinical Sci. 78:155–158) have shown an abundance of peripheral-type benzodiazepine receptors (PBR) compared to normal tissue. Moreover, a 12-fold increase in PBR density relative to normal parenchyma, was found in human brain glioma or astrocytoma (Cornu, P. et al. (1992) Acta Neurochir. 199:146–152). The authors suggested that PBR densities may reflect the proliferative activity of the receptor in these tissues. Recently, the involvement of PBR in cell proliferation was further shown (Neary, J. T. et al. (1995) Brain Research 675:27–30; Miettinen, H. et al. (1995) Cancer Research 55:2691–2695), and its expression of human astrocytic tumors was found to be associated with tumor malignancy and proliferative index (Miettinen, H. et al. supra; Alho, H. (1994) Cell Growth Different. 5:1005–1014). Further studies have shown that PBR receptors are abundant in human glioblastomas (Broaddus, W. C., et al., Brain Research, Vol. 518:199–208, 1990; and Pappata, S., et al., J. Nuclear Med., 32:1608–1610, 1991).
PBR is an 18-kDa protein discovered as a class of binding sites for benzodiazepines distinct from the GABA neurotransmitter receptor (Papadopoulos, V. (1993) Endocr. Rev. 14:222–240). PBR are extremely abundant in steroidogenic cells and found primarily on outer mitochondrial membranes (Anholt, R. et al. (1986) J. Biol. Chem. 261:576–583). PBR is thought to be part of the multimeric complex composed of the 18-kDa isoquinoline-binding protein and the 34-kDa pore-forming voltage-dependent anion channel protein, preferentially located on the outer/inner mitochondrial membrane contact sites (McEnery, M. W. et al. Proc. Natl. Acad. Sci. U.S.A. 89:3170–3174; Garnier, M. et al. (1994) Mol. Pharmacol. 45:201–211; Papadopoulos, V. et al. (1994) Mol. Cel. Endocr. 104:R5–R9). Drug ligands of PBR, upon binding to the receptor, stimulate steroid synthesis in steroidogenic cells in vitro (Papadopoulos, V. et al. (1990) J. Biol. Chem. 265:3772–3779; Ritta, M. N. et al. (1989) Neuroendocrinology 49:262–266; Barnea, E. R. et al. (1989) Mol. Cell Endocr. 64:155–159; Amsterdam, A. and Suh, B. S. (1991) Endocrinology 128:503–510; Yanagibashi, K. et al. (1989) J. Biochem. (Tokyo) 106:1026–1029). Likewise, in vivo studies showed that high affinity PBR ligands increase steroid plasma levels in hypophysectomized rats (Papadopoulos V. et al (1997) Steroids 62:21–28). Further in vitro studies on isolated mitochondria provided evidence that PBR ligands, drug ligands, or the endogenous PBR ligand, the polypeptide diazepam binding inhibitor (BDI) (Papadopoulos, V. et al. (1997) Steroids 62:21–28), stimulate pregnenolone formation by increasing the rate of cholesterol transfer from the outer to the inner mitochondrial membrane (Krueger, K. E. and Papadopoulos, V. (1990) J. Biol. Chem 265:15015–15022; Yanagibashi, K. et al. (1988) Endocrinology 123: 2075–2082; Besman, M. J. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:4897–4901; Papadopoulos, V. et al. (1991) Endocrinology 129:1481–1488).
Based on the amino acid sequence of the 18-kDa PBR, a three dimensional model was developed (Papadopoulos, V. (1996) In: The Leydig Cell. Payne, A. H. et al. (eds) Cache River Press, IL, pp. 596–628). This model was shown to accomodate a cholesterol molecule and function as a channel, supporting the role of PBR in cholesterol transport. Recently we demonstrated the role of PBR in steroidogenesis by generating PBR negative cells by homologous recombination (Papadopoulos, V. et al. (1997) J. Biol. Chem. 272:32129–32135) that failed to produce steroids. However, addition of the hydrosoluble analogue of cholesterol, 22R-hydroxycholesterol, recovered steroid production by these cells, indicating that the cholesterol transport mechanism was impaired. Further cholesterol transport experiments in bacteria expressing the 18-kDa PBR protein provided definitive evidence for a function as a cholesterol channel/transporter (Li and Papadopoulos, V. et al., (1998) Endocrinology).
We hypothesized that the peripheral-type benzodiazepine receptor is part of the changes in cellular and molecular functions that account for the increased aggressive behavior in cancer, and we chose to examine this hypothesis in human breast cancer. Breast cancer is the most common neoplasm and the leading cause of cancer-related deaths for women in most developing countries (Lippman, M. E. (1993) Science 259:631–632), affecting nearly 184,000 women, with over 46,000 deaths annually in the U.S. alone (American Cancer Society, 1996). Human breast cells are unlike brain and gonadal cells and cannot produce steroids, but like many other cells in the body, are able to metabolize steroids.
Increased PBR expression correlates with increased aggressive behavior of tumor cells. Invasive tumors invade and grow locally but they do not metastasize. However, the aggressive tumors have the ability to invade and metastasize through the blood vessels to different places of the human body. Tumor metastasis into vital organs (such as lungs) is the most common cause of death.
The correlation between high levels of expression of PBR and metastatic potential in for human breast cancer is shown in copending U.S. application Ser. No. 09/047,652 filed Mar. 25, 1998, in which Vassilios Papadopoulos of the instant application is a co-inventor. However, due to the involvement of PBR in cell proliferation, and the expression of PBR in all cells, it is likely that this correlation would exist for other solid tumors and cancers such as prostate cancer, colon cancer, brain tumors, and tumors in steroid producing tissues such as gonadal tumors, to name a few.